1. Field of the Invention
This invention relates to the formation of microcylinders or microtubules from phospholipids with the rational control of wall thickness (i.e. the number of bilayers) of microcylinders.
2. Description of the Previously Published Art
Multibilayer lipid microstructures have been made by cooling from multilamellar liposomes in water, and from solutions of ethanol and other alcohols. The resulting multibilayer tubules have typically a broad distribution of bilayers with a median of about 10 bilayers. Single bilayer tubules have been prepared by cooling from solutions of water and methanol. Single bilayer tubules, however, are extremely fragile.
U.S. Pat. No. 4,990,291 discloses forming lipid microcylinders from a thermal cycling process in what is basically a lipid/water system. After purification and drying of a diacetylenic phospholipid the tubule is formed by first hydrating the lipid at about 10° C. above its endothermic transition point. Then the lipid mixture is cooled slowly at a rate not to exceed 1° C. per minute, preferably not greater than 0.5° C. per min to a formation temperature 1° to 10° below the lipids' exothermic transition temperature also known as the gel phase transition. The solution is held at the formation temperature for between 30 minutes and 2 hours, most preferably 1 hour. Once the tubule structures are formed they are stable as long as the tubule structures are not heated above the endothermic transition temperature. If desired, the tubule structures can be polymerized by any of the well-known means to a permanent tubule form. The tubules formed are usually extremely straight hollow cylinders of approximately 0.5 micrometer diameter and 5 to 100 micrometers in length. These tubules can be used in a vast variety of ways. The tubules can be used to hold materials in a manner well known for lipid vesicles described in the patent. In addition, the tubules can be coated with metals as described in U.S. Pat. No. 4,911,981. This U.S. Pat. No. 4,990,291 patent does not use methanol or methanol/water solutions, or describe any process of crystallization from a mixed alcohol water system.
U.S. Pat. No. 4,887,501 discloses forming lipid microstructure from a mixed solvent system to produce helix or cylinder microstructures. The first step is to add a lipid to a lipid solvating organic solvent. Then a predetermined amount of water is added to the solvent/lipid mixture and the solution is allowed to sit for a predetermined amount of time and at a predetermined temperature. The temperature is preferably maintained about 10–30° C. below the melting point of the lipid as defined in excess water. A broad range of solvents are disclosed and the concentration of the lipid in the organic solvent lipid solution is typically preselected to be less than about 2 mg/ml.
The most preferred organic solvents are relatively polar organic solvents such as tetrahydrofuran, chloroform, and alcohols and polyols, such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, propylene glycol, ethylene glycol and mixtures of these. In the 14 examples the only alcohols used are ethanol and isopropanol. There is no discussion of the number of bilayers in the tubule wall. In Example 4 the diameters range from 0.2 to 3.0 microns. This reference does not describe a method to yield a uniform microtubule dispersion at high yield. It also does not teach rational control of bilayer numbers or any method of obtaining the same. The lipid is first dissolved in the solvent and then the water added. When practiced as described, the direct result of the addition of water is the immediate formation of lipid structures from the dissolved lipid in the presence of local concentrations of water. This leads to a very large number of liposomes, bilayer ribbons and sheets as well as micelles formed in addition to the desired microtubules. Such materials degrade the sample, decrease the yield of microtubules and thus diminish the degree of control that may otherwise be realized.
U.S. Pat. No. 4,887,501 further teaches that a range of bilayers resulted from formation in a mixed solvent system, and that variation in the solvent concentrations as well as the concentration of the lipid in relation to the mixed solvent had little effect on the number of bilayers, and that in contrast the number of bilayers were effected to a greater extent by the hydrocarbon chain length in the lipids used to form the microtubules.
To date the yield and morphology of the microtubules has been difficult to reproduce on a rational basis, and especially conversion rates have been poor. This problem exists in both systems of thermal cycling and mixed solvents. It is almost impossible to obtain conversion rates that are economically viable with thermal cycling, and in addition there is no correlation between concentration and aspect ratio or yield. Further the presence of a very large number of non-cylindrical lipid structures makes it very difficult if not impossible to process the resultant structures into a homogenous cylindrical product. In the solvent methodology it is imperative that thermal and chemical mixing (including the heat of mixing of the alcohol and water) be minimized. Any mixing once the tubules have formed leads to differential shear and thus mechanical disruption of the high aspect ratio microcylinders and also leads to birdnesting which can be thought of as a tubule logjam. Such inconsistent morphology results in microcylinders which may vary from 4 to 20 or more bilayers in a single batch. The inner diameters remain about 0.5 microns with the outer diameters varying as a result of the number of bilayers.
These multiple-walled cylinders have the further problem that the yield (and thus the costs) of actual microcylinders from an initial concentration of lipid may be up to 500% lower than if the same amount of lipid was used to form microcylinders with a fixed, lower number of bilayers such as a double bilayer wall per structure.
Although one might desire to make smaller tubules from an economic point of view, the literature does not teach one how this can be done and especially how it can be done efficiently. It also does not teach the minimum number of bilayers that are needed for a strong product.
B. R. Ratna et al in “Effect of alcohol chain length on tubule formation in 1,2-bis(10,12-tricosadiynol)-sn-glycero-3-phosphocholine,” Chem. Phy. Lipids. 63, 47 (1992) studied the volume fractions of alcohols from which tubule formation was observed. For the methanol/water system they found the range to be 65/25–90/10. For lower fractions the lipid precipitates out in an amorphous form whereas on the higher alcohol side of the window, the lipid remains in solution even at room temperature. The authors also measured the number of bilayers in the tubules grown from different alcohols. They found the number of bilayers constituting the wall of the tubule is independent of the alcohol/water ratio. However, the number is found to be strongly dependent on the chain length of the alcohol. They studied methanol, ethanol and 1-propanol. For methanol and using an 85/15 methanol/water system, 95% of the tubules grown were made of a single bilayer and the remaining 5% being made from two or three bilayers. There is no teaching that the number of bilayers could be controlled at two to form a more robust structure, and in fact teaches away from utilization of methanol to make multi-layered structures. The paper clearly indicates that there is no way using its reaction conditions that more than one bilayer tubules can be formed from methanol-water solutions. When the alcohol was changed to ethanol or 1-propanol the wall thickness as well as its variance increase considerable. Samples grown in both of these longer chain solvents have an average of 6–7 bilayers with a standard deviation of 3 bilayers. Thus the literature has taught that a methanol/water system basically produces a microtubule with a wall having only a single bilayer.
These tubules produced from methanol, however, which normally have the single bilayer break very easily and can not be used for metalization and subsequent commercial applications.
G. Nounesis et al in “Melting of Phospholipid Tubules,” Phy. Rev. Lett. 76, 3650 (1996) describe the morphological transformations and the bilayer phase transformation of DC8,9GPC tubules in methanol/water solutions. They note that at a lipid concentration, ρ, less than 2 mg/cm3 the majority, ˜95%, of the tubules formed have single bilayer walls. With ρ>4 mg/cm3,—most of the tubules have from two to four bilayers in the walls. This discussion illustrates the common knowledge that the wall produced in methanol/water system is generally a single bilayer. Although at a higher lipid concentration wall is produced having more than a single bilayer, there is no suggestion that just a two bilayer can be produced since they report that the microtubules have from 2 to 4 bilayers.
Previous work with ethanol/water systems in both thermal cycling process and the mixed solvent system has indicated that the median number of bilayers in any population of microcylinders is about 10, and many have been observed having up to 20 or more. For the case of the smallest median wall size of approximately 10 bilayers for the ethanol system, the number of microstructures that could possibly form is reduced by at least 500% at a minimum over a more desired system where the average wall size is only two bilayers.
3. Objects of the Invention
It is an object of this invention to provide for the rational control of the number of lipid bilayers that comprise lipid microstructures such as lipid tubules.
It is a further object of this invention to provide the formation of robust microtubules which can be processed for further treatment without disruption of the microstructure.
It is a further object of this invention to provide lipid microstructures which have greater than a single bilayer microtubule so that they will be sufficiently robust to allow for continued processing such as metallization, inclusion in polymer or ceramic matrixes, or exposed to in vivo as well as in vitro environments.
It is a further object of this invention to provide lipid microstructures which have just two bilayers so as to form robust tubules with the minimum amount of lipid material.
It is a further object of this invention to form lipid microstructures with an aspect ratio of the tubules which is greater obtained than from previous methods.
It is a further object of this invention to form lipid microstructures having a much narrower range of diameters, due to the regularity of the number of bilayers.
It is a further object of this invention to provide for a process for the rational control of the number of lipid bilayers comprising the walls of microcylinders.
It is a further object of this invention to provide a process to produce lipid microstructures which have greater than a single bilayer microtubule so that they will be sufficiently robust to allow for continued processing.
It is a further object of this invention to form robust lipid microstructures where the number of bilayers is less than in other methods yet at least two bilayers so there are more individual tubules formed from the same weight of lipid.
It is a further object of this invention to increase the yield of individual lipid microstructure from a initial concentration of lipid monomer.
It is a further object of this invention to provide a substantial cost reduction in the process of forming lipid microstructures due to the use of more efficient production with increased overall yield.
It is a further object of this invention to provide greater conversion of lipid to microcylinders such that the yield is >90% conversion.
It is a further object of this invention to provide the ability to control the number of bilayers when forming lipid microstructures to be within a narrow range.
It is a further object of this invention to provide a process to make lipid microcylinders of predetermined morphology from a mixed methanol/water system.
It is a further object of this invention to provide a means of thermally cycling a lipid/solvent system whereby the aspect ratio of the microcylinders may be controlled.
It is an object of this invention to provide a method in which microcylinders formed from lipids may be produced in bulk by a rational process, whereby the number of lipid bilayers that comprise lipid microcylinders may be predetermined to the optimum number of two, with a conversion rate of >98% of the lipid utilized, thus offering a very large increase in the numbers of such structures per volume of lipid and alcohol, and thus reducing costs up to 100 to 500%.
These and further objects of the invention will become apparent as the description of the invention proceeds.